The potential for human and ecological toxicity associated with nanomaterials and ultrafine particles is a growing area of investigation as more nanomaterials and products are developed and brought into commercial use. To date, few nanotoxicology studies have addressed the effects of nanomaterials in a variety of organisms and environments. However, the existing research raises some concerns about the safety of nanomaterials and has led to increased interest in studying the toxicity of nanomaterials for use in risk assessment and protection of human health and the environment. A new field of nanotoxicology has been developed to investigate the possibility of harmful effects due to exposure to nanomaterials [3
]. Nanotoxicology also encompasses the proper characterization of nanomaterials used in toxicity studies. Characterization has been important in differentiating between naturally occurring forms of nanomaterials, nano-scale byproducts of natural or chemical processes, and manufactured (engineered) nanomaterials. Because of the wide differences in properties among nanomaterials, each of these types of nanoparticles can elicit its own unique biological or ecological responses. As a result, different types of nanomaterials must be categorized, characterized, and studied separately, although certain concepts of nanotoxicology based on the small size, likely apply to all nanomaterials.
As materials reach the nanoscale, they often no longer display the same reactivity as the bulk compound. For example, even a traditionally inert bulk compound, such as gold, may elicit a biological response when it is introduced as a nanomaterial [4
]. New approaches for testing and new ways of thinking about current materials are necessary to provide safe workplaces, products, and environments as the manufacturing of nanomaterials and products increases and, as a result, exposure to nanomaterials increases. The diverse routes of exposure, including inhalation, dermal uptake, ingestion, and injection, can present unique toxicological outcomes that vary with the physicochemical properties of the nanoparticles in question.
The earliest studies investigating the toxicity of nanoparticles focused on atmospheric exposure of humans and environmentally relevant species to heterogeneous mixtures of environmentally produced ultrafine particulate matter (having a diameter <100 nm). These studies examined pulmonary toxicity associated with particulate matter deposition in the respiratory tract of target organisms [5
]. Epidemiological assessments of the effects of urban air pollution exposure focusing on particulate matter produced as a byproduct of combustion events, such as automobile exhaust and other sources of urban air pollution, showed a link in test populations between morbidity and mortality and the amount of particulate matter [16
]. Some researchers have found an increased risk of childhood and adult asthma correlated to environmental exposure to ultrafine particulate matter in urban air [20
]. However, other research does not indicate the same correlation [23
Laboratory-based studies have investigated the effects of a large range of ultrafine materials through in vivo
exposures using various animal models as well as cell-culture-based in vitro
experiments. To date, animal studies routinely show an increase in pulmonary inflammation, oxidative stress, and distal organ involvement upon respiratory exposure to inhaled or implanted ultrafine particulate matter [7
]. Tissue and cell culture analysis have also supported the physiological response seen in whole animal models and yielded data pointing to an increased incidence of oxidative stress, inflammatory cytokine production, and apoptosis in response to exposure to ultrafine particles [31
]. These studies have also yielded information on gene expression and cell signaling pathways that are activated in response to exposure to a variety of ultrafine particle species ranging from carbon-based combustion products to transition metals. Polytetrafluoroethylene fumes in indoor air pollution are nano-sized particles, highly toxic to rats [38
]. They elicit a severe inflammatory response at low inhaled particle mass concentrations, suggestive of an oxidative injury [39
In contrast to the heterogeneous ultrafine materials produced incidentally by combustion or friction, manufactured nanomaterials can be synthesized in highly homogenous forms of desired sizes and shapes (e.g., spheres, fibers, tubes, rings, planes). Limited research on manufactured nanomaterials has investigated the interrelationship between the size, shape, and dose of a material and its biological effects, and whether a unique toxicological profile may be observed for these different properties within biological models.
Typically, the biological activity of particles increases as the particle size decreases. Smaller particles occupy less volume, resulting in a larger number of particles with a greater surface area per unit mass and increased potential for biological interaction [42
]. Recent studies have begun to categorize the biological response elicited by various nanomaterials both in the ecosystem and in mammalian systems. Although most current research has focused on the effect of nanomaterials in mammalian systems, some recent studies have shown the potential of nanomaterials to elicit a phytotoxic response in the ecosystem. In the case of alumina nanoparticles, one of the US market leaders for nano-sized materials, 99.6% pure nanoparticles with an average particle size of 13 nm were shown to cause root growth inhibition in five plant species [46
Toxicological studies of fibrous and tubular nanostructures have shown that at extremely high doses these materials are associated with fibrotic lung responses and result in inflammation and an increased risk of carcinogenesis. Single-walled carbon nanotubes (SWCNT) have been shown to inhibit the proliferation of kidney cells in cell culture by inducing cell apoptosis and decreasing cellular adhesive ability. In addition, they cause inflammation in the lung upon instillation [26
]. Multi-walled carbon nanotubes (MWCNT) are persistent in the deep lung after inhalation and, once there, are able to induce both inflammatory and fibrotic reactions [47
Dermal exposure to MWCNT has been modeled through cell culture and points to the nanoparticles' ability to localize within and initiate an irritation response in target epithelial cells [50
]. Proteomic analysis conducted in human epidermal keratinocytes exposed to MWCNT showed both increased and decreased expression of many proteins relative to controls. These protein alterations suggested dysregulation of intermediate filament expression, cell cycle inhibition, altered vesicular trafficking/exocytosis and membrane scaffold protein down-regulation [50
]. In addition, gene expression profiling was conducted on human epidermal keratinocytes exposed to SWCNT that showed a similar profile to alpha-quartz or silica. Also, genes not previously associated with these particulates before from structural protein and cytokine families were significantly expressed [52
]. Dosing keratinocytes and bronchial epithelial cells in vitro
with SWCNT has been shown to result in increases in markers of oxidative stress [50
Charge properties and the ability of carbon nanoparticles to affect the integrity of the blood-brain barrier as well as exhibit chemical effects within the brain have also been studied. Nanoparticles can overcome this physical and electrostatic barrier to the brain. In addition, high concentrations of anionic nanoparticles and cationic nanoparticles are capable of disrupting the integrity of the blood-brain barrier. The brain uptake rates of anionic nanoparticles at lower concentrations were greater than those of neutral or cationic formulations at the same concentrations. This work suggests that neutral nanoparticles and low concentration anionic nanoparticles can serve as carrier molecules providing chemicals direct access to the brain and that cationic nanoparticles have an immediate toxic effect at the blood-brain barrier [55
Tests with uncoated, water soluble, colloidal C60
fullerenes have shown that redox-active, lipophilic carbon nanoparticles are capable of producing oxidative damage in the brains of aquatic species [55
]. The bactericidal potential of C60
fullerenes was also observed in these experiments. This property of fullerenes has possible ecological ramifications and is being explored as a potential source of new antimicrobial agents [57
Oxidative stress as a common mechanism for cell damage induced by nano- and ultrafine particles is well documented; fullerenes are model compounds for producing superoxide. A wide range of nanomaterial species have been shown to create reactive oxygen species both in vivo
and in vitro
. Species which have been shown to induce free radical damage include the C60
fullerenes, quantum dots, and carbon nanotubes [30
]. Nanoparticles of various sizes and chemical compositions are able to preferentially localize in mitochondria where they induce major structural damage and can contribute to oxidative stress [65
Quantum dots (QDs) such as CdSe QDs have been introduced as new fluorophores for use in bioimaging. When conjugated with antibodies, they are used for immunostaining due to their bright, photostable fluorescence.
To date, there is not sufficient analysis of the toxicity of quantum dots in the literature, but some current studies point to issues of concern when these nanomaterials are introduced into biological systems. Recently published research indicates that there is a range of concentrations where quantum dots used in bioimaging have the potential to decrease cell viability, or even cause cell death, thus suggesting that further toxicological evaluation is urgently needed [67
]. While it is well known that bulk cadmium selenide (CdSe) is cytotoxic, it has been suggested that CdSe quantum dots are cytocompatible, and safe for use in whole animal studies. This postulate is based in part on the use of protecting groups around the CdSe core of the quantum dot. These coatings have been shown to be protective, but their long-term stability has not been evaluated thoroughly. Recent studies exploring the cytotoxicity of CdSe-core quantum dots in primary hepatocytes as a liver model found that these quantum dots were acutely toxic under certain conditions. The cytotoxicity correlates with the liberation of free Cd2+
ions due to deterioration of the CdSe lattice. These data suggest that quantum dots can be rendered nontoxic initially for in vivo
use when appropriately coated. However, the research also highlights the need to further explore the long-term stability of the coatings used, both in vivo
and exposed to environmental conditions [69
The range of approaches and methods used to reach conclusions regarding the effects of manufactured nanomaterials and ultrafine particles has led to different results. This inconsistency indicates a need for standardized tests in order to get comparable results in screening nanomaterials for potential adverse effects. As the field of nanotoxicology continues to grow, standard toxicology tests will aid those entering the field and allow for better comparisons and conclusions in determining the toxic effects of nanomaterials.